Glycosaminoglycans vary in accumulation along the neuraxis during spinal neurulation in the mouse embryo

We have utilized the method of whole embryo culture for metabolic labeling of mouse embryos with [3H]glucosamine during closure of neural folds at the posterior neuropore (27- to 29-somite stage). Accumulations of newly synthesized glycopeptides, lactosaminoglycans, hyaluronate, and sulfated glycosaminoglycans (GAG) were assessed by ion-exchange chromatography of glycoconjugates isolated from labeled embryos. Accumulation of hyaluronate and sulfated GAG was greatest in the posterior neuropore and decreased progressively toward the hindbrain where neurulation was already complete. Hyaluronate comprised a progressively smaller proportion of total newly synthesized glycoconjugate from the posterior neuropore toward the cranial region and glycopeptides showed the opposite trend. Sulfated GAG and lactosaminoglycans showed no consistent differences in relative abundance along the neuraxis. Autoradiographic analysis of newly synthesized glycoconjugates revealed especially heavy incorporation into developing basement membranes, beneath the neuroepithelium and around the notochord, in the posterior neuropore and recently closed neural tube regions, but not at more cranial levels of the neuraxis. Predigestion of sections with a specific hyaluronidase showed a significant quantity of this glycoconjugate to be hyaluronate. These results are consistent with a role for neuroepithelial and notochordal basement membrane hyaluronate in spinal neurulation.     

Accumulation of basement membrane-associated hyaluronate is reduced in the posterior neuropore region of mutant (curly tail) mouse embryos developing spinal neural tube defects

We investigated the accumulation of newly synthesized glycoconjugates during spinal neurulation in mutant curly tail mouse embryos, a proportion of which develop lower spinal neural-tube defects (NTD). Embryos undergoing closure of the posterior neuropore (27- to 29-somite stage) were labeled in vitro with [3H]glucosamine, and [3H]glycoconjugates were analyzed by ion-exchange chromatography. Mutant embryos undergoing normal spinal neurulation exhibited a pattern of glycoconjugate accumulation closely similar to that observed for nonmutant embryos (Copp and Bernfield, 1988, Dev. Biol. 130, 573-582). Mutant embryos developing spinal NTD accumulated reduced amounts of [3H]hyaluronate specifically in the posterior neuropore region. Other embryonic regions and other glycoconjugates appeared unaffected by the developmental abnormality. Autoradiographic analysis of labeled curly tail embryos confirmed that [3H]hyaluronate accumulates in reduced amounts in the posterior neuropore region and indicated that this reduction is mainly localized to the site of developing basement membranes, beneath the neuroepithelium and around the notochord. Accumulation of [3H]hyaluronate in the interstitial mesenchymal matrix of the posterior neuropore region is not consistently affected in embryos developing spinal NTD. These results provide support for a role for basement-membrane hyaluronate in lower spinal neurulation.     

Time-lapse photographic study of neural tube closure defects caused by xylocaine in the chick

Sequential changes in the morphology of early chick embryos were, for the first time, photographically recorded. Embryos were explanted at stage 8 (four-somite) or 9- (six-somite) of development using New's technique and grown in nutrient medium (thin albumen) with or without a teratologic dose (200 micrograms/ml) of xylocaine. They were photographed using a Nikon Diaphot inverted microscope equipped with both phase-contrast optics and photomicrographic accessories maintained in an incubator. It was found, among other things, that a characteristic neural tube closure defect often seen in the midbrain and anterior portion of the hindbrain of xylocaine (200 micrograms/ml)-treated chick embryos was a consequence of failure of the neural tube to withstand the tension generated by the rapidly expanding cephalic region, which occurred, regardless of the stage at explanation, when corresponding control embryos had advanced to stage 10+ (11-somite) of development.     

Vital dye analysis of cranial neural crest cell migration in the mouse embryo.

The spatial and temporal aspects of cranial neural crest cell migration in the mouse are poorly understood because of technical limitations. No reliable cell markers are available and vital staining of embryos in culture has had limited success because they develop normally for only 24 hours. Here, we circumvent these problems by combining vital dye labelling with exo utero embryological techniques. To define better the nature of cranial neural crest cell migration in the mouse embryo, premigratory cranial neural crest cells were labelled by injecting DiI into the amniotic cavity on embryonic day 8. Embryos, allowed to develop an additional 1 to 5 days exo utero in the mother before analysis, showed distinct and characteristic patterns of cranial neural crest cell migration at the different axial levels. Neural crest cells arising at the level of the forebrain migrated ventrally in a contiguous stream through the mesenchyme between the eye and the diencephalon. In the region of the midbrain, the cells migrated ventrolaterally as dispersed cells through the mesenchyme bordered by the lateral surface of the mesencephalon and the ectoderm. At the level of the hindbrain, neural crest cells migrated ventrolaterally in three subectodermal streams that were segmentally distributed. Each stream extended from the dorsal portion of the neural tube into the distal portion of the adjacent branchial arch. The order in which cranial neural crest cells populate their derivatives was determined by labelling embryos at different stages of development. Cranial neural crest cells populated their derivatives in a ventral-to-dorsal order, similar to the pattern observed at trunk levels. In order to confirm and extend the findings obtained with exo utero embryos, DiI (1,1-dioctadecyl-3,3,3',3'-tetramethylindo-carbocyanine perchlorate) was applied focally to the neural folds of embryos, which were then cultured for 24 hours. Because the culture technique permitted increased control of the timing and location of the DiI injection, it was possible to determine the duration of cranial neural crest cell emigration from the neural tube. Cranial neural crest cell emigration from the neural folds was completed by the 11-somite stage in the region of the rostral hindbrain, the 14-somite stage in the regions of the midbrain and caudal hindbrain and not until the 16-somite stage in the region of the forebrain. At each level, the time between the earliest and latest neural crest cells to emigrate from the neural tube appeared to be 9 hours.(ABSTRACT TRUNCATED AT 400 WORDS)     

Neural Tube Closure in Mouse Whole Embryo Culture

Genetic mouse models are an important tool in the study of mammalian neural tube closure (Gray & Ross, 2009; Ross, 2010). However, the study of mouse embryos in utero is limited by our inability to directly pharmacologically manipulate the embryos in isolation from the effects of maternal metabolism on the reagent of interest. Whether using a small molecule, recombinant protein, or siRNA, delivery of these substances to the mother, through the diet or by injection will subject these unstable compounds to a variety of bodily defenses that could prevent them from reaching the embryo. Investigations in cultures of whole embryos can be used to separate maternal from intrinsic fetal effects on development.Here, we present a method for culturing mouse embryos using highly enriched media in a roller incubator apparatus that allows for normal neural tube closure after dissection (Crockett, 1990). Once in culture, embryos can be manipulated using conventional in vitro techniques that would not otherwise be possible if the embryos were still in utero. Embryo siblings can be collected at various time points to study different aspects of neurulation, occurring from E7-7.5 (neural plate formation, just prior to the initiation of neurulation) to E9.5-10 (at the conclusion of cranial fold and caudal neuropore closure, Kaufman, 1992). In this protocol, we demonstrate our method for dissecting embryos at timepoints that are optimal for the study of cranial neurulation. Embryos will be dissected at E8.5 (approx. 10-12 somities), after the initiation of neural tube closure but prior to embryo turning and cranial neural fold closure, and maintained in culture till E10 (26-28 somities), when cranial neurulation should be complete.     

Development of dorsal-ventral polarity in the optic vesicle and its presumptive role in eye morphogenesis as shown by embryonic transplantation and in ovo explant culturing

Dorsal and ventral specification in the early optic vesicle appears to play a crucial role in the proper development of the eye. In the present study, we performed embryonic transplantation and organ culturing of the chick optic vesicle in order to investigate how the dorsal-ventral (D-V) polarity is established in the optic vesicle and what role this polarity plays in proper eye development. The left optic vesicle was cut and transplanted inversely in the right eye cavity of host chick embryos. This method ensured that the D-V polarity was reversed while the anteroposterior axis remained normal. The results showed that the location of the choroid fissure was altered from the normal (ventral) to ectopic positions as the embryonic stage of transplantation progressed from 6 to 18 somites. At the same time, the shape of the optic vesicle and the expression patterns of Pax2 and Tbx5, marker genes for ventral and dorsal regions of the optic vesicle, respectively, changed concomitantly in a similar way. The crucial period was between the 8- and 14-somite stages, and during this period the polarity seemed to be gradually determined. In ovo explant culturing of the optic vesicle showed that the D-V polarity and choroid fissure formation were already specified by the 10-somite stage. These results indicate that the D-V polarity of the optic vesicle is established gradually between 8- and 14-somite stages under the influence of signals derived from the midline portion of the forebrain. The presumptive signal(s) appeared to be transmitted from proximal to distal regions within the optic vesicle. A severe anomaly was observed in the development of optic vesicles reversely transplanted around the 10-somite stage: the optic cup formation was disturbed and subsequently the neural retina and pigment epithelium did not develop normally. We concluded that establishment of the D-V polarity in the optic vesicle plays an essential role in the patterning and differentiation of the neural retina and pigment epithelium.     

SEM observations of the neural fold associated with neurulation in the rat

The topography of the ectoderm was examined by scanning electron microscopy during neurulation in rat embryos at stage 24 (somites 8-11). A zone of altered cell morphology was observed along the crest of neural folds. This zone was located between the presumptive neural tube and the surface ectoderm and exhibited numerous rounded cell blebs, immediately prior to fusion between the folds. It is suggested that the observed surface alterations may reflect a change in the properties of the altered cell which correlate with initial adhesion between the folds.     

Immunohistochemical localization of chondroitin and heparan sulfate proteoglycans in pre-spina bifida splotch mouse embryos.

The splotch (Sp) mutation on mouse chromosome I is a genetic model for the neural tube defects spina bifida and exencephaly. Embryos carrying Sp or its allele splotch-delayed (Spd), have been shown to have delays in neural tube closure, and neural crest cell emigration, as well as a reduction in extracellular space around the neural tube. Pre-spina bifida Sp and Spd embryos have abnormalities of notochord, mesoderm and neuroepithelial development. Chondroitin sulphate proteoglycans (CSPG) and heparan sulfate proteoglycans (HSPG) have been shown to play essential roles during neural tube closure and neural crest cell emigration and migration and thus might well be affected by the splotch mutation. Therefore, the effects of Sp and Spd on the temporal and spatial distributions of CSPG and HSPG were studied in pre-spina bifida embryos cytogenetically identified as Sp/Sp (Spd/Spd), Sp/ + (Spd/ +) or +/+. Immunohistochemical localization of CSPG by means of the CS-56 monoclonal antibody showed that in Sp/Sp head sections, the neuroepithelial basement membranes stained more intensely at 5-, 10-, and 15-somite stages, whereas similar staining was observed at 16- and 19-somite stages compared with matched +/+ sections. In caudal sections Sp/Sp again showed a more intense stain for CSPG in the neuroepithelial basement membranes in all sections (except one comparison, in which staining was similar) from embryos of 14-, 15-, 16-, and 19-somite stages, compared to matched +/+ sections. Heterozygotes did not differ consistently from the mutant or the normal (+/+) embryos in CS-56 stain intensity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Neural crest development in the Xenopus laevis embryo, studied by interspecific transplantation and scanning electron microscopy

The Xenopus borealis quinacrine marker and scanning electron microscopy have been used to study the appearance, migration, and homing of neural crest cells in the embryo of Xenopus. The analysis shows that the primordium of the neural crest develops from the nervous layer of the ectoderm and consists of three segments at early neurula stages. This primordium is located in the lateral halves of the neural folds behind the prospective eye vesicles. The histological and experimental evidence shows that the neural crest cells also originate from the medial portion of the neural folds. The neural crest segments in the cephalic region start to migrate just before the closure of the neural tube. Isotopic and isochronic unilateral grafts of X. borealis neural crest into X. laevis embryos were performed in order to map the fate of the cranial crest segments and the vagal-truncal neural crest. The analysis of the X. laevis host embryos shows that the mandibular crest segment contributes to the lower jaw (Meckel's cartilage), quadrate, and ethmoid-trabecular cartilages, as well as to the ganglionic and Schwann cells of the trigeminus nerve, the connective tissues, the mesenchymal and choroid layers of the eye, and the cornea. The hyoid crest segment is located in the ceratohyal cartilage and in ganglia VII and VIII. The branchial crest segment migrates from the caudal part of the otic vesicle and divides into two portions which contribute to the cartilages of the gills. The vagal-truncal neural crest starts to migrate later at stage 25. It migrates by means of the vagus complex in a ventral direction and penetrates into the splanchnic layer of the digestive tract. The trunk neural crest cells disperse into three different pathways which differ from those of the avian embryo at this level.     

Studies on the mechanisms of neurulation in the chick: interrelationship of contractile proteins, microfilaments, and the shape of neuroepithelial cells

Electron microscopy and indirect immunofluorescence were employed to correlate the distribution patterns of major contractile proteins (actin and myosin) with 1) the organizational state of microfilaments, 2) the apical cell surface topography, 3) the shape of the neuroepithelial cells, and 4) the degree of bending of the neuroepithelium during neurulation in chick embryos at Hamburger and Hamilton stages 5-10 of development. Both actin and myosin are present at these developmental stages and colocalize in the neural plate as well as in later phases of neurulation. During elevation of neural folds, actin- and myosin-specific fluorescence is always most intense in regions where the greatest degree of bending of the neuroepithelium takes place [e.g., the midline of the V-shaped neuroepithelium (early neural fold stage) and the midlateral walls of the "C"-shaped neuroepithelium (mid-neural-fold stage)]. This intense fluorescence coincides with 1) a particularly dense packing of microfilaments and 2) highly constricted cell apices. After neural folds make contact, there is an overall reduction in both the intensity of apical fluorescence and the thickness of apical microfilament bundles, especially in the roof and floor of the neural tube. The remaining fluorescence in the contact area is apparently related to cellular movements during fusion of neural folds.     

The developmental fate of the cephalic mesoderm in quail-chick chimeras.

The developmental fate of the cephalic paraxial and prechordal mesoderm at the late neurula stage (3-somite) in the avian embryo has been investigated by using the isotopic, isochronic substitution technique between quail and chick embryos. The territories involved in the operation were especially tiny and the size of the transplants was of about 150 by 50 to 60 microns. At that stage, the neural crest cells have not yet started migrating and the fate of mesodermal cells exclusively was under scrutiny. The prechordal mesoderm was found to give rise to the following ocular muscles: musculus rectus ventralis and medialis and musculus oblicus ventralis. The paraxial mesoderm was separated in two longitudinal bands: one median, lying upon the cephalic vesicles (median paraxial mesoderm--MPM); one lateral, lying upon the foregut (lateral paraxial mesoderm--LPM). The former yields the three other ocular muscles, contributes to mesencephalic meninges and has essentially skeletogenic potencies. It contributes to the corpus sphenoid bone, the orbitosphenoid bone and the otic capsules; the rest of the facial skeleton is of neural crest origin. At 3-somite stage, MPM is represented by a few cells only. The LPM is more abundant at that stage and has essentially myogenic potencies with also some contribution to connective tissue. However, most of the connective cells associated with the facial and hypobranchial muscles are of neural crest origin. The more important result of this work was to show that the cephalic mesoderm does not form dermis. This function is taken over by neural crest cells, which form both the skeleton and dermis of the face. If one draws a parallel between the so-called "somitomeres" of the head and the trunk somites, it appears that skeletogenic potencies are reduced in the former, which in contrast have kept their myogenic capacities, whilst the formation of skeleton and dermis has been essentially taken over by the neural crest in the course of evolution of the vertebrate head.     

Aberrant convergence of the neural folds in the mouse mutant vl.

Progressive changes in the dorsolateral angles (DA) and ventral angle (VA) during elevation and convergence of the caudal neural folds were morphometrically analyzed in normal and dysraphic abnormal embryos of the mouse mutant vacuolated lens (vl), and correlations with the configuration of microfilaments in the apices of neuroepithelial cells were made by means of ultrastructural cytochemistry. In 22-28 somite stage abnormal (vl/vl) embryos, the DA and VA are larger than those in their normal counterparts at each comparable level of the caudal neural folds, suggesting that defective convergence involves both the DA and VA in this mutant. In 30-35 somite stage abnormal embryos, the VA is likewise larger than that in normal embryos in which the neural folds have converged and closed; however, the DAs are much smaller, indicating that a medial collapse of the dorsal ends of the neural folds may occur secondary to the closure failure. At the DA, the ultrastructural configuration of microfilaments is similar in abnormal and normal embryos in terms of their circumferential arrangement around the perimeters of the neuroepithelial cell apices. In abnormal embryos, however, the bundles of microfilaments are more delicate and less prominent than in normal embryos; thus it is possible that a quantitative and/or functional deficiency in these elements may be involved in the failure of the abnormal neuroepithelium to bend properly during convergence of the neural folds.     

Effects of antibodies against N-cadherin and N-CAM on the cranial neural crest and neural tube.

We have examined the distribution and function of the defined cell adhesion molecules, N-cadherin and N-CAM, in the emigration of cranial neural crest cells from the neural tube in vivo. By immunocytochemical analysis, both N-cadherin and N-CAM were detected on the cranial neural folds prior to neural tube closure. After closure of the neural tube, presumptive cranial neural crest cells within the dorsal aspect of the neural tube had bright N-CAM and weak N-cadherin immunoreactivity. By the 10- to 11-somite stage, N-cadherin was prominent on all neural tube cells with the exception of the dorsal-most cells, which had little or no detectable immunoreactivity. N-CAM, but not N-cadherin, was observed on some migrating neural crest cells after their departure from the cranial neural tube. To examine the functional significance of these molecules, perturbation experiments were performed by injecting antibodies against N-CAM or N-cadherin into the cranial mesenchyme adjacent to the midbrain. Fab' fragments or whole IgGs of monoclonal and polyclonal antibodies against N-CAM caused abnormalities in the cranial neural tube and neural crest. Predominantly observed defects included neural crest cells in ectopic locations, both within and external to the neural tube, and mildly deformed neural tubes containing some dissociating cells. A monoclonal antibody against N-cadherin also disrupted cranial development, with the major defect being grossly distorted neural tubes and some ectopic neural crest cells outside of the neural tube. In contrast, nonblocking N-CAM antibodies and control IgGs had few effects. Embryos appeared to be sensitive to the N-CAM and N-cadherin antibodies for a limited developmental period from the neural fold to the 9-somite stage, with older embryos no longer displaying defects after antibody injection. These results suggest that the cell adhesion molecules N-CAM and N-cadherin are important for the normal integrity of the cranial neural tube and for the emigration of neural crest cells. Because cell-matrix interactions also are required for proper emigration of cranial neural crest cells, the results suggest that the balance between cell-cell and cell-matrix adhesion may be critical for this process.     

An ultrastructural examination of the role of cell membrane surface coat material during neurulation

Data from neural crest cultures indicate that cell surface coat material (CSM) is directly involved in cellular migration and events surrounding differentiation. To investigate whether the CSM also has a morphogenetic role, embryos of the amphibian Ambystoma maculatum were examined ultrastructurally throughout the stages of neurulation. Segments of the neural axis were fixed in glutaraldehyde-containing Alcian blue 8GX, which reportedly enhances preservation of CSM, and were postfixed in OsO4 containing 1 percent lanthanum nitrate, which stains the CSM. The medial groove formed by the appearance of the neural ridges contains a large amount of CSM and numerous vesicles coated with lanthanum-positive material. In contrast, the lateral ridge surfaces are covered by a small amount of uniformly distributed CSM and a paucity of vesicles. As the ridges begin to fold there is a progressive increase in the amount of CSM within the presumptive neural tube region. Further convergence of the neural folds is accompanied by an increase of CSM at their leading edges. As the folds approximate each other, lanthanum-positive material physically bridges the gap. However, as the apposing tissue actually abuts to form the neural tube, no CSM is observed in the remaining interspace. The specific distribution and sequential accumulation of cell CSM during the events of neurulation strongly suggest its direct participation in the morphogenetic process.     

Cytochemical evidence for the neural crest origin of mammalian ultimobranchial C cells

Summary The cells of the neural crest have APUD properties at an early stage of devel opment (72 hours in the chick embryo). The FIF procedure provides a cytochemical means for their distinction.Using mouse embryos from mothers injected, intraperitoneally, 1 hr before removal, with l-DOPA (100 mg/kg), the peripheral stream of neural crest cells was clearly identifiable at the 7-somite stage (7–8 days). At the 10-somite stage (8–9 days) the cells were observed to invade the lateral processes of the foregut, and the foregut itself. A particularly high concentration of fluorescent APUD cells was observed in the anterior portion of the IVth pharyngeal pouch, destined to become the ultimobranchial body.At the 14-somite stage (11–12 days) the developing ultimobranchial body still contains fluorescent cells of neural crest origin.The implications of these findings on the question of the origin of the entire APUD series of endocrine polypeptide cells is discussed.     

Developmentally regulated expression and functional role of alpha 7 integrin in the chick embryo

Integrin alpha 7 beta 1 is a specific cellular receptor for laminin. In the present work, we studied the distribution pattern of the alpha 7 subunit by immunofluorescence and immunoprecipitation and the role of the integrin by blocking antibodies in early chick embryos. alpha 7 immunoreactivity was first detectable in the neural plate during neural furrow formation (stage HH5, early neurula, Hamburger & Hamilton 1951) and its expression was upregulated in the neural folds during primary neurulation. The alpha 7 expression domain spanned the entire neural tube by stage HH8 (4 somites), and was then downregulated and confined to the neuroepithelial cells in the germinal region near the lumen and the ventrolateral margins of the neural tube in embryos by the onset of stage HH17 (29 somites). Expression of alpha 7 in the neural tube was transient suggesting that alpha 7 functions during neural tube closure and axon guidance and may not be required for neuronal differentiation or for the maintenance of the differentiated cell types. alpha 7 immunoreactivity was strong in the newly formed epithelial somites, although this expression was restricted only to the myotome in the mature somites. The most intense alpha 7 immunoreactivity was detectable in the paired heart primordia and the endoderm apposing the heart primordia in embryos at stage HH8. In the developing heart, alpha 7 immunoreactivity was: (i) intense in the myocardium; (ii) milder in the endocardial cushions of the ventricle; (iii) intense in the sinus venosus; (iv) distinct in the associated blood vessels; and (v) undetectable in the dorsal mesocardium of embryos at stage HH17. Inhibition of function of alpha 7 by blocking antibodies showed that alpha 7 integrin-laminin signaling may play a critical role in tissue organization of the neural plate and neural tube closure, in tissue morphogenesis of the heart tube but not in the directional migration of pre-cardiac cells, and in somite epithelialization but not in segment formation in presomitic mesoderm. In embryos treated with alpha 7 antibody, the formation of median somites in place of a notochord was intriguing and suggested that alpha 7 integrin-laminin signaling may have played a role in segment re-specification in the mesoderm.     

Neural plate morphogenesis during mouse neurulation is regulated by antagonism of Bmp signalling

Dorsolateral bending of the neural plate, an undifferentiated pseudostratified epithelium, is essential for neural tube closure in the mouse spinal region. If dorsolateral bending fails, spina bifida results. In the present study, we investigated the molecular signals that regulate the formation of dorsolateral hinge points (DLHPs). We show that Bmp2 expression correlates with upper spinal neurulation (in which DLHPs are absent); that Bmp2-null embryos exhibit premature, exaggerated DLHPs; and that the local release of Bmp2 inhibits neural fold bending. Therefore, Bmp signalling is necessary and sufficient to inhibit DLHPs. By contrast, the Bmp antagonist noggin is expressed dorsally in neural folds containing DLHPs, noggin-null embryos show markedly reduced dorsolateral bending and local release of noggin stimulates bending. Hence, Bmp antagonism is both necessary and sufficient to induce dorsolateral bending. The local release of Shh suppresses dorsal noggin expression, explaining the absence of DLHPs at high spinal levels, where notochordal expression of Shh is strong. DLHPs ;break through' at low spinal levels, where Shh expression is weaker. Zic2 mutant embryos fail to express Bmp antagonists dorsally and lack DLHPs, developing severe spina bifida. Our findings reveal a molecular mechanism based on antagonism of Bmp signalling that underlies the regulation of DLHP formation during mouse spinal neural tube closure.     

Retinaldehyde dehydrogenase 2 (RALDH2)-mediated retinoic acid synthesis regulates early mouse embryonic forebrain development by controlling FGF and sonic hedgehog signaling

Although retinoic acid (RA) has been implicated as one of the diffusible signals regulating forebrain development, patterning of the forebrain has not been analyzed in detail in knockout mouse mutants deficient in embryonic RA synthesis. We show that the retinaldehyde dehydrogenase 2 (RALDH2) enzyme is responsible for RA synthesis in the mouse craniofacial region and forebrain between the 8- and 15-somite stages. Raldh2-/- knockout embryos exhibit defective morphogenesis of various forebrain derivatives, including the ventral diencephalon, the optic and telencephalic vesicles. These defects are preceded by regionally decreased cell proliferation in the neuroepithelium, correlating with abnormally low D-cyclin gene expression. Increases in cell death also contribute to the morphological deficiencies at later stages. Molecular analyses reveal abnormally low levels of FGF signaling in the craniofacial region, and impaired sonic hedgehog signaling in the ventral diencephalon. Expression levels of several regulators of diencephalic, telencephalic and optic development therefore cannot be maintained. These results unveil crucial roles of RA during early mouse forebrain development, which may involve the regulation of the expansion of neural progenitor cells through a crosstalk with FGF and sonic hedgehog signaling pathways.